The present invention relates to the field of microscopic sample analysis, and specifically relates to a device for preparing thin microscopic samples of liquids.
Microscopic examination of blood films is an important part of the hematologic evaluation. Today, three methods for preparing thin blood films are in use, namely the xe2x80x9cwedge-slide methodxe2x80x9d, the xe2x80x9cspinner methodxe2x80x9d and the xe2x80x9ccoverglass methodxe2x80x9d (see, e.g. xe2x80x9cClinical Diagnosis and Management by Laboratory Methodsxe2x80x9d, Nineteenth Edition, 1996, edited by John B. Henry). In the wedge-slide method, a drop of blood is placed onto a slide that is on a flat surface. A second (spreader) slide is pressed at an angle of 30 to 45 degrees against the first slide and moved along the first slide, which results in the formation of a moderately thin blood film that is then dried in air. The quality of the generated blood smear will strongly depend on the personal skill level of the technician. To overcome the need for highly skilled personnel, manual apparatus (U.S. Pat. No. 4,494,479 to Drury) and automated apparatus (U.S. Pat. No. 4,407,843 to Sasaki; U.S. Pat. No. 3,683,850 to Grabhorn; U.S. Pat. No. 3,880,111 to Levine et al.; U.S. Pat. No. 4,392,450 to Prevo; WO 9,641,148 to Levine et al.) for executing the wedge-slide method have been proposed.
In addition to being time-consuming, the physical action of the spreader slide tends to distort the morphology of many of the cells. In view of this, an alternative method for the preparation of blood samples has been proposed (e.g., U.S. Pat. No. 5,549,750 to Kelley) wherein a drop of blood is disposed onto a slide which is then spun to create a monolayer of randomly distributed red blood cells. It has been found, however, that drying of the red blood cells produces undesirable types of distortions, particularly a loss of central pallor for many of the red cells as they dry. It is not entirely clear what causes these shape changes, but they apparently are caused by surface tension, charges and/or drying effects. To inhibit cell morphology distortions from occurring during drying, it has been proposed to preserve the morphologies by applying fixing agents after forming the monolayer, but prior to drying (U.S. Pat. No. 4,209,548 to Bacus; U.S. Pat. No. 4,483,882 to Saunders).
The wedge-slide method as well as the spinner method require relatively complex apparatus and involve time-consuming procedures. A simpler way to produce blood films is the coverglass method where two quadratic coverglasses are being used. A first glass, with a drop of blood attached to the center of the underside, is placed crosswise on a second glass so that the corners appear as an eight-pointed star. If the drop is not too large and if the glasses are perfectly clean, the blood will spread out evenly and quickly in a thin layer between the two surfaces.
After spreading stops, the two glasses are pulled apart on a plane parallel to their surfaces. The two blood films are then dried in air.
While the coverglass method does not require auxiliary apparatus, the quality of the blood smears will again depend strongly on the level of skill of the technician performing the procedure. Moreover, executing the method includes increased risk because the thin pieces of glass that contain the blood sample may break during the separation step. And, finally, drying the blood films may cause changes in the cell morphology.
Consequently, there exists a need for a simple method and device for producing thin blood samples for microscopic analysis that does not require auxiliary apparatus, provides monolayers of red blood cells, and avoids changes in the cell morphology.
It is an objective of the present invention to provide a method and device for producing thin samples of liquids for microscopic analysis, and preferably, thin samples of blood, that does not require auxiliary apparatus; provides, in the instance of blood, monolayers of red blood cells; and avoids changes in the cell morphology.
According to the present invention, the above objective is achieved by disposing a drop of a liquid sample, such as, for example, blood, onto a microscope slide near to its center, by arranging spacers on the slide outside of the slide""s center, by positioning a flexible coverglass onto the spacers, by applying a downward force to, for example, the center of the flexible coverglass so that the coverglass touches the blood, and by suspending the application of said force. In the moment the coverglass touches the drop of sample, the sample spreads outwards and adhesion forces hold the flexible coverglass down so that a very thin layer of liquid is formed.
According to the invention, it is important that the spread sample fills the space between the slide and the coverglass only partially. This can be accomplished by selecting the height of the spacers accordingly. Preferably, the spacers can have a height of from about 10 micrometers (xcexcm) to about 3000 xcexcm, and more preferably from about 50 xcexcm to about 200 xcexcm. The coverglass is held in a stable position due to an equilibrium state between said adhesion forces and counter forces generated as a consequence of coverglass bending.
It has been found that a liquid blood sample prepared according to the present invention comprises a central area which contains plasma but no red blood cells. This region is surrounded by a wide ring containing huge numbers of isolated red blood cells in a well-defined monolayer arrangement. The ring in turn is surrounded by an even wider belt that contains red blood cells in Rouleaux formation (blood cell aggregation) where the length of the Rouleaux blocks increases with increasing distance from the center. This kind of blood sample preparation does not result in morphology changes as observed in the wedge-slide method or during drying of blood films in the open air. Due to the coverglass bending, regions of varying sample thickness are available for examination.
Of course, as noted above, the method and device for sample preparation according to the present invention is not limited to blood samples. However, in the following detailed description, the invention will be disclosed in more detail, using blood as a preferred example.